Nuchal edema (NE) is a tissue swelling on the back of the neck of fetus due to an abnormal fluid accumulation during human pregnancy and has been associated with fetuses having chromosomal aneuploidy. NE can be clinically diagnosed by ultrasound and the measurement of NE, also called nuchal translucency measurement, has been widely accepted as a standard prenatal screening method to assess the risk of the fetus having chromosomal abnormalities. Studies show that more than 80% of NE fetuses carry chromosomal abnormalities such as Down syndrome. Although NE has been reported to be associated with disturbed development of the lymphatic system, the molecular basis of NE is currently unknown. In this proposal, we provide our preliminary evidence that Notch signal is dysregulated in the lymphatic system of human trisomy 21 Down syndrome fetuses and their mouse model, trisomy 16 mice embryos that exhibit NE. Previous studies show that Notch signal plays an essential role in specifying arterial cell fate. Consistently, we found that Notch can reprogram lymphatic endothelial cells (LECs) to abort the normal lymphatic differentiation program and adopt the arterial endothelial cell fate. Interestingly, a similar abnormal arterialization has been reported to occur in the venous compartment of knockout mice for the nuclear receptor COUP-TFII, a finding that indicates the key role of COUP-TFII in Notch repression in the veins. We also discovered that two Down syndrome-associated genes DSCR1 and Dyrk1a, when ectopically expressed in primary LECs, down-regulate COUP-TFII. Based on these data, we now build a novel model for the molecular basis of NE: Increased gene dosage of DSCR1 and Dyrk1a results in down-regulation of COUP- TFII and subsequent activation of Notch signal in LECs of Down syndrome fetuses, and the dysregulated Notch signal induces a pathological arterialization of the developing lymphatics, which fails embryonic tissue fluid homeostasis, causing NE. In this proposal, we propose to further dissect the molecular mechanism underlying the pathological arterialization of developing lymphatics. The outcome of our proposed studies will not only advance our current understanding of the molecular mechanism underlying the developmental programs for the arterio-venous-lymphatic endothelial cell fate specification, but also provide important insights into other vascular defects during human development.